Abbreviations footnote: HSPG, heparan sulfate proteoglycan; HS, heparan sulfate; FGF, fibroblast growth factor; FGF2, fibroblast growth factor 2, also known as bFGF; FGF4, fibroblast growth factor 4, also known as eFGF; GlcNR6Sase, GlcNR 6-O exosulfatase; FGFR1, FGF receptor 1; QSulf1, quail Sulf1; ERK, extracellular signaling regulated kinase.
HSPGs are extracellular matrix glycoproteins that regulate cell surface signaling during embryogenesis and pathophysiology of diseases. HSPGs include a protein core coupled to several covalently linked HS chains that bind to signaling molecules. HS chains are composed of 50-200 disaccharide repeats of uronic acid and glucosamine residues that are selectively sulfated at 2-O position of uronic acid and 6-O, 3-O, and N positions of glucosamine residues. The sulfation patterns of HS are further regulated to create highly sulfated and undersulfated domains along the length of the HS chain, leading to structural heterogeneity.
Sulfation of HS chains is required for developmental signaling processes in embryonic cells. Loss of HS sulfation in Drosophilia sulfateless and slalom mutants leads to defects in Wingless (Wg) and FGF or Wg and Hedgehog (Hh) signaling, respectively, and to defects in tissue patterning in the embryo. Further, sulfation deficiencies at individual positions within the HS disaccharide unit also cause signaling defects, as revealed in studies of mutations that disrupt HS biosynthesis. For example, mice with a gene-trap mutation in Hs2st, a key HS 2-O-sulfotransferase, lack 2-O sulfated uronic acid and exhibit lethal kidney agenesis due to defects in multiple signaling processes. RNAi inhibition of Drosophila HS 6-O-sulfotransferase gene expression reduces FGF signaling activity and disrupts the primary branching of the tracheal system. Consistent with these genetic studies, treatment of cells in culture with chlorate to inhibit HS sulfation results in defects in BMP, Wnt and FGF signaling. HS sulfation, therefore, plays important roles in multiple signaling activities in embryos. However, the biological mechanisms for regulating the sulfation states of HS in embryos and the biochemical roles of specific HS sulfate groups in the control of ligand activity and/or receptor interactions are not well understood.
Recently, a family of HS 6-O endosulfatases have been identified that modify HS 6-O sulfation and developmental signaling in embryonic cells, providing a new class of evolutionarily conserved regulators of HS sulfation. A second, closely related family member Sulf2 has been identified in mammals and birds. Sulf1 exhibits structural and enzymatic features distinct from known glucosamine 6-O sulfatases (GlcNR6Sase), which are lysosomal exosulfatases that catalyze the hydrolysis of terminal 6-O sulfo groups during HS degradation. In contrast, Sulf1 is secreted through the Golgi and is docked on the cell surface through its distinctive hydrophilic domain, and Sulf1 functions as a 6-O endosulfatase, with substrate specificity for trisulfated IdoA2S-GlcNS6S disaccharide units of HS/heparin. The avian ortholog, QSulf1, is required for Wnt-dependent gene expression in muscle progenitor cells of the quail embryo. QSulf1 activity remodels the 6-O sulfation states of cell surface HSPGs and decreases the binding affinity between Wnt ligand and HS.
Biochemical and crystallographic studies show that HS sulfation is required for FGF ligand-receptor interactions and FGF signaling. HS chains containing trisulfated disaccharide units greatly promote FGF2-FGFR1 binding and signaling, although FGF2 can bind to FGFR1 in the absence of HS in cell binding assays and in crystallographic studies. Among the sulfate groups on HS, sulfation at 6-O position of glucosamine residues is required for FGF2-FGFR1 and FGF4-FGFR1 interactions and signaling. Although distinct sequences and sulfation patterns in HS chains are required for FGF ligand and receptor binding, 6-O sulfation of HS is crucial for FGF signaling activity.
FGFs and FGFRs (FGF receptors) play critical roles in many developmental and disease processes including angiogenesis and cancer. Sulf1 is a cell surface 6-O HS endosulfatase expressed in embryonic cell lineages controlled by multiple signaling pathways including FGF, and recent studies reveal that the human Sulf1 ortholog, HSulf1, can down-regulate FGF-dependent ERK kinase activity in human cancer cells. The ability to regulate FGF-controlled developmental process in embryos through a more detailed understanding of these pathways would represent a significant advance in the art.